PB1771: OVERCOMING RESISTANCE TO TYROSINE KINASE INHIBITORS IN MYELOID LEUKEMIA CELLS THROUGH ENHANCED FERROPTOSIS
Authors: Chih-Fang Chang Chien, Yu-Hsuan Fu, Chi-Yang Tseng, Ching-Ying Kuo, Liang-In Lin
Journal: HemaSphere (2023)

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: We previously established Molm13-XR leukemia cell line from parental Molm13, which is resistant to multiple tyrosine kinase inhibitors (TKIs) including sorafenib, cabozantinib, and quizartinib. Whole-transcriptome profiles and GSEA revealed that both “fatty acid derivative biosynthesis” and “ferroptosis” pathways were enriched in Molm13-XR. Among them, the upregulated genes associated with defense-related ferroptosis, implying that alternative ferroptosis inducers could overcome this drug resistance. Ferroptosis is a programmed death that requires iron and involves fatty acid metabolism and oxidative phosphorylation. Malignant cells with aberrant redox-related enzymes or amino acid transporters usually do not undergo ferroptosis even if they are subjected to an environment enrich in catalytic iron and lipid peroxides. Aims: To investigate the feasibility and therapeutic strategy of ferroptosis inducer to overcome the TKIs-resistance of Molm13-XR cells. Methods: TKI-resistant Molm13-XR myeloid leukemia cell line was used. Erastin, sulfasalazine, iFSP1, and RSL3 were used to induce ferroptosis. Flow cytometry was applied to analyze cell cycle, mitochondria ROS and C11 BODIPY. Results: Further extracting the variations of ferroptosis-related genes from the transcriptomic data, we found that compared to that in parental cells, the ferroptosis-associated genes, SLC7A11 (3.24x, p< 0.0001), AIFM2 (FSP1) (82.3, p< 0.0001x), AKR1C1 (10.44x, p= 0.0399), AKR1C2 (8.68x, p< 0.0001), GCH1 (3.39x, p= 0.0077), ACSL1 (2.13x, p< 0.0001), and SAT1 (1.87x, p= 0.0233) were significantly higher in Molm13-XR cells. Q-RT-PCR confirmed the higher expression of these genes in Molm13-XR cells. Further immunoblotting also demonstrated their protein status. System Xc-, consisting of the SLC7A11 and SLC3A2 subunits, functions as a cystine/glutamate antiporter. The increased SLC711A could increase the uptake of cystine, forming GSH to escape from ferroptosis. The IC50 of two system Xc- inhibitors, erastin and sulfasalazine in Molm13-XR cells were up to 7.0 μM and 457.7 μM, respectively, indicating other cystine transporter could compensate this defect of ferroptosis pathway. Another upregulated ferroptosis-related gene, ALFM2, encodes ferroptosis suppressor protein 1 (FSP1). FSP1 functions as an NADPH-dependent CoQ oxidoreductase to prevent lipid peroxidation and ferroptosis. iFSP1 (inhibitor of FSP1) was a potent, selective glutathione-independent inhibitor of FSP. However, the IC50 of iFSP1 was also up to 9.3 μM, indicating it is not proper ferroptosis inducer for Molm13-XR cells. Transcriptomic analysis revealed no significant difference in GPX4 gene expression between parental and resistant cells, suggesting the of GPX4-associated ferroptosis pathway was still intact. RSL3 undergoes ferroptosis by directly deactivating GPX4. The IC50 of RSL3 was 104.3 nM, indicating the feasibility of RSL3 for ferroptosis in Molm13-XR cells. We found that RSL3 could potently inhibit the function of GPX4 in inducing lipid peroxidation, as seen using C11 BOCIPY analysis. In addition, RSL3 could significantly increase oxidative stress in the mitochondria and induce cell cycle G0/G1 arrest in XR cells. Summary/Conclusion: Our results suggest that ferroptosis was defective in TKIs-resistant cells and that intact GPX4, but not SLC7A11 and AIFM2, is a suitable therapeutic target and RSL3 is such an appropriate agent. Keywords: RNA-seq, Drug resistance, Tyrosine kinase inhibitor, Acute myeloid leukemia